High performance liquid chromatography (HPLC) has become one of the most useful analytical tools in a modern chemical laboratory. It is particularly useful for the room temperature separation and analysis of very small amounts of organic or biochemical compounds. Most commonly, in HPLC a spectrophotometer is used to detect the presence of the analyte, since most organic compounds will absorb radiation in the range of about 190 nm to 800 nm, particularly in the UV range of about 190 nm to 350 nm.
Although HPLC is a powerful analytical tool for the separation and detection of low amounts of organic or biochemical compounds, sufficiently low detection limits are still not currently available for many analytes including conjugated aromatic and non-aromatic compounds, such as polycyclic aromatic compounds, including substituted naphthalenes and anthracenes; anthraquinones; biphenyls, polychlorinated biphenyls; aromatic and non-aromatic nitrogen, oxygen and sulfur containing heterocyclic compounds, conjugated non-aromatic compounds such as biacetal and its derivatives where very low detection limits are desired. For example, the process of this invention is particularly suitable for the detection of very low amounts of pharmaceuticals in body fluids or toxic substances in the environment.
Phosphorescence or fluorescence has been considered to improve the sensitivity of the detection limit of HPLC (1). However, many UV or VIS absorbing compounds have low or negligible fluorescence quantum yields (2), or only phosphoresce at low temperatures or in organized media.
A photochemical method of transforming UV absorbing compounds such as Clobazam and Fenbendazole into fluorescent substances has been reported (1). However, this approach requires the analyte compound to be transformable to fluorescent compounds, and few organic compounds meet this requirement. Other photochemical methods using a post-column photochemical reactor to enhance detection limits have also been reported (3,13,14). These include a colorimetric method of determining nitroso compounds by photohydrolysis to nitrites; photolytic decomposition of halogen, or nitrogen or sulfur containing compounds; and the photoreduction of anthraquinone derivatives by glycosides and saccharides. However, these methods are generally applicable only to very specific types of compounds.
Several approaches to the use of phosphorescence in HPLC have been suggested including micelle stabilized room temperature phosphorescence (4), sensitized room temperature phosphorescence (5) and phosphorescence quenching (6). However, all of these approaches require the careful removal of virtually all dissolved oxygen from the solvents which are used for dissolving the samples containing the analyte(s) and the mobile phase. This necessity of rigorous oxygen removal is a severe disadvantage. Furthermore, none of these approaches have resulted in substantial improvements in the detection limits when compared to conventional UV absorption.
Many efforts have been made to study the photochemical reaction of organic compounds in the presence of oxygen (2, 7, 8, 9, 10, 11, 14). The research efforts indicate that molecular oxygen reversibly quenches the excited singlet and triplet states of many organic molecules, i.e. fluorescent or phosphorescent organic molecules.
It is also known that the interaction of molecular oxygen with electronically excited molecules lead to the formation of a metastable excited singlet state oxygen (3). Metastable singlet oxygen apparently is the responsible intermediate in certain photooxygenation reactions of two classes of organic compounds: (A) those that have the structural element of cis-1,3-dienes and those that are polycyclic aromatic compounds and heterocyclic compounds such as furans; or (B) olefins containing allylic hydrogen atoms (7).
Compounds which are electronically excitable, i.e. compounds which absorb radiation at a wavelength greater than 240 nm to produce triplet state molecules with a quantum yield greater than about 0.05, with a triplet state lifetime greater than about 10.sup.-6 seconds, and have triplet state energies above 22.5 kcal/mole have been found to be useful to catalyze the photooxygenation reaction.
It has been reported that xanthene dyes and rose bengal when excited by light energy can transfer the energy to oxygen molecules to form singlet oxygen which then reacts with 1,3-diphenylisobenzofuran or 2,5-dimethylfuran (7) and (8). Many other organic compounds have been shown to be good sensitizers for singlet oxygen formation (7).
A solid-state peroxyoxalate chemiluminescence detection of hydrogen peroxide generated in a post column reaction using HPLC has been reported recently (8). Hydrogen peroxide was generated in a post column photochemical reaction from the analyte quinone separated and eluted by HPLC. The amount of hydrogen peroxide was detected using solid state peroxyoxalate energy transfer chemiluminescence of bis-2,4,6-trichlorophenyl oxalate. However, using this method the detection limits of quinone derivatives have not been found to be improved from that of using UV absorption.
Post column photochemical reactors have also been described (1, 2, 9, 13, 14). Uihlein and Schwab described the use of a post column photochemical reaction chamber made from polytetrafluoroethylene (PTFE) tubing. The chamber is made by knitting thin PTFE tubing with 0.5 mm I.D. into a strand of loops which were then supported around a light source by being woven on a star shaped device. The knitted reaction chamber, without the use of a column, showed band broadening of HETP of 1.9 cm (1).
A reaction chamber knitted into a cylinder from thin PTFE tubing to fit around a fluorescent lamp tube and a pyrex tube was also described (13,14). The use of a crocheted post column chemical reactor was mentioned by Poulsen and Birks et al. (9).
None of these developments have led to a method for improving the detection limits of HPLC by the use of the photocatalyzed reactions of substituted furans, bilirubin, chlorophylls, substituted pyrroles, substituted imidazoles, and substituted olefins.
The object of the present invention is to develop a method of improving the detection limits of HPLC for a class of radiation absorbing organic compounds without the need of rigorous removal of oxygen dissolved in the solvents.
A second object is to improve the detectability of most radiation absorbing organic compounds in HPLC by at least an order of magnitude.
A further object is to improve the detectability of most radiation absorbing organic compounds in HPLC by a simple and convenient post column photochemical reaction.